The value of laboratory animal health monitoring to the performance of reliable biomedical research has been well established. Although serologic assays have provided a cornerstone for the detection of viruses and several bacterial species in laboratory rodents, there is an increasing need to develop other methods to detect these agents. High-throughput assays that detect nucleic acid sequences specific for individual and/or groups of infectious agents would provide an ideal supplement to serologic testing and could be used to evaluate whole animals and biomaterials such as transplantable tumors and cell lines. Traditional polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) assays have been developed for multiple infectious agents and have demonstrated the usefulness of this type of methodology in rodent diagnostics. However, currently available PCR and RT-PCR assays generally target one infectious agent, vary greatly in their thermocycling parameters, and require post-PCR processing for the detection of PCR products, all of which preclude high-throughput evaluation of samples for multiple agents. Fluorogenic 5' prime nuclease assays (FFPNA) have been developed that amplify and detect specific DNA and RNA sequences without the need for post-PCR processing. This technology lends itself to the development of high-throughput assays that can be used to efficiently monitor for multiple infectious agents from a variety of biological specimens. The overall goal of this project is to develop FFPNA for the direct identification of infectious agents known to naturally infect laboratory rodents and/or contaminate biomaterials that are inoculated into rodents. The specific aims of this project are to develop genus- and/or species-specific FFPNA for most viruses and several non-viral agents that infect laboratory rodents, and to evaluate these assays as an alternative to rodent antibody production (RAP) testing and as an adjunct methodology for laboratory rodent health monitoring. FFPNA will greatly enhance the diagnostic capabilities for routine health monitoring of laboratory rodents and will provide a viable, cost-effective alternative for RAP testing.